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Identification of transcripts associated with the acquisition of superior freezing tolerance in recurrently-selected populations of alfalfa

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Abstract

The identification of transcripts that vary during the acquisition of superior tolerance to environmental stress contribute to the understanding of the molecular bases of adaptation to adverse growth conditions. SRAP-cDNA was used to identify genes that were differentially expressed between plants of alfalfa (Medicago sativa L.) that were non-acclimated (NA) or hardened to a non-lethal freezing temperature (HF). Search of polymorphism was performed within the cultivar Apica (ATF0) and population ATF5 obtained after five cycles of recurrent selection for superior tolerance to freezing (TF) within ATF0. Analysis of bulked cDNA from 50 non-acclimated and 50 cold-acclimated plants in each population identified transcripts that vary in abundance in response to acclimation to sub-zero temperature and recurrent selection. Sequencing of purified fragments revealed significant homologies with genes with key physiological functions that are distributed across the Medicago truncatula genome. Regulatory genes including receptor-like kinases, a phytochrome-interacting factor and a phosphoinositide phosphatase are among potential candidates involved in the improvement of freezing tolerance by RS. RT-qPCR analysis of samples from populations recurrently selected within the cultivars Apica and Evolution confirmed the common cold-induced responses in unrelated genetic backgrounds. Our results show that random amplification of cDNA in combination with bulk segregant analysis of pooled samples from recurrently selected populations is an effective strategy to identify functional sequences putatively associated with superior acclimation to environmental stress in open-pollinated species.

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References

  1. Agarwal M, Shrivastava N, Padh H (2008) Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Rep 27(4):617–631. https://doi.org/10.1007/s00299-008-0507-z

  2. Arora R, Rowland LJ, Panta GR (1997) Chill-responsive dehydrins in blueberry: are they associated with cold hardiness or dormancy transitions? Physiol Plant 101(1):8–16. https://doi.org/10.1111/j.1399-3054.1997.tb01813.x

  3. Bélanger G, Castonguay Y, Bertrand A, Dhont C, Rochette P, Couture L, Drapeau R, Mongrain D, Chalifour F-P, Michaud R (2006) Winter damage to perennial forage crops in eastern Canada: causes, mitigation, and prediction. Can J Plant Sci 86:33–47

  4. Brummer EC (2004) Applying genomics to alfalfa breeding programs. Crop Sci 44(6):1904–1907

  5. Castonguay Y, Nadeau P, Laberge S (1993) Freezing tolerance and alteration of translatable mRNAs in alfalfa (Medicago sativa L.) hardened at subzero temperatures. Plant Cell Physiol 34(1):31–38

  6. Castonguay Y, Laberge S, Brummer EC, Volenec JJ (2006) Alfalfa winter hardiness: a research retrospective and integrated perspective. In: Donald LS (ed) Advances in agronomy, vol 90. Academic Press, Cambridge, pp 203–265. https://doi.org/10.1016/S0065-2113(06)90006-6

  7. Castonguay Y, Michaud R, Nadeau P, Bertrand A (2009) An indoor screening method for improvement of freezing tolerance in alfalfa. Crop Sci 49(3):809. https://doi.org/10.2135/cropsci2008.09.0539

  8. Castonguay Y, Cloutier J, Bertrand A, Michaud R, Laberge S (2010) SRAP polymorphisms associated with superior freezing tolerance in alfalfa (Medicago sativa spp. sativa). Theor Appl Genet 120(8):1611–1619. https://doi.org/10.1007/s00122-010-1280-2

  9. Castonguay Y, Bertrand A, Michaud R, Laberge S (2011) Cold-induced biochemical and molecular changes in alfalfa populations selectively improved for freezing tolerance. Crop Sci 51(5):2132–2144. https://doi.org/10.2135/cropsci2011.02.0060

  10. Castonguay Y, Dubé M-P, Cloutier J, Michaud R, Bertrand A, Laberge S (2012) Intron-length polymorphism identifies a Y2K4 dehydrin variant linked to superior freezing tolerance in alfalfa. Theor Appl Genet 124(5):809–819. https://doi.org/10.1007/s00122-011-1735-0

  11. Castonguay Y, Michaud J, Dubé M-P (2015) Reference genes for RT-qPCR analysis of environmentally and developmentally regulated gene expression in alfalfa. Am J Plant Sci 6(01):132

  12. Crowe JH, Hoekstra FA, Crowe LM (1992) Anhydrobiosis. Annu Rev Physiol 54(1):579–599

  13. de Lorenzo L, Merchan F, Laporte P, Thompson R, Clarke J, Sousa C, Crespi M (2009) A novel plant leucine-rich repeat receptor kinase regulates the response of Medicago truncatula roots to salt stress. Plant Cell 21(2):668–680. https://doi.org/10.1105/tpc.108.059576

  14. Dube MP, Castonguay Y, Cloutier J, Michaud J, Bertrand A (2013) Characterization of two novel cold-inducible K3 dehydrin genes from alfalfa (Medicago sativa spp. sativa L.). Theor Appl Genet 126(3):823–835. https://doi.org/10.1007/s00122-012-2020-6

  15. Espevig T, DaCosta M, Hoffman L, Aamlid TS, Tronsmo AM, Clarke BB, Huang B (2011) Freezing tolerance and carbohydrate changes of two Agrostis species during cold acclimation. Crop Sci 51(3):1188–1197

  16. Falke K, Flachenecker C, Melchinger A, Piepho H-P, Maurer H, Frisch M (2007) Temporal changes in allele frequencies in two European F 2 flint maize populations under modified recurrent full-sib selection. Theor Appl Genet 114(5):765–776

  17. Fan X, Naz M, Fan X, Xuan W, Miller AJ, Xu G (2017) Plant nitrate transporters: from gene function to application. J Exp Bot 68(10):2463–2475

  18. Gao Y, Jiang W, Dai Y, Xiao N, Zhang C, Li H, Lu Y, Wu M, Tao X, Deng D (2015) A maize phytochrome-interacting factor 3 improves drought and salt stress tolerance in rice. Plant Mol Biol 87(4–5):413–428

  19. Gui Q, Wang J, Xu Y, Wang J (2009) Expression changes of duplicated genes in allotetraploids of < i>Brassica </i > detected by SRAP-cDNA technique. Mol Biol 43(1):1–7. https://doi.org/10.1134/s0026893309010014

  20. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series pp 95–98

  21. Herman EM, Rotter K, Premakumar R, Elwinger G, Bae H, Ehler-King L, Chen S, Livingston DP 3rd (2006) Additional freeze hardiness in wheat acquired by exposure to − 3 °C is associated with extensive physiological, morphological, and molecular changes. J Exp Bot 57(14):3601–3618. https://doi.org/10.1093/jxb/erl111

  22. Hundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genom 9(1):118

  23. Jiang B, Shi Y, Zhang X, Xin X, Qi L, Guo H, Li J, Yang S (2017) PIF3 is a negative regulator of the CBF pathway and freezing tolerance in Arabidopsis. Proc Natl Acad Sci 114(32):E6695–E6702

  24. Jung J-H, Domijan M, Klose C, Biswas S, Ezer D, Gao M, Khattak AK, Box MS, Charoensawan V, Cortijo S (2016) Phytochromes function as thermosensors in Arabidopsis. Science 354(6314):886–889

  25. Le MQ, Engelsberger WR, Hincha DK (2008) Natural genetic variation in acclimation capacity at sub-zero temperatures after cold acclimation at 4°C in different Arabidopsis thaliana accessions. Cryobiology 57(2):104–112. https://doi.org/10.1016/j.cryobiol.2008.06.004

  26. Le MQ, Pagter M, Hincha DK (2015) Global changes in gene expression, assayed by microarray hybridization and quantitative RT-PCR, during acclimation of three Arabidopsis thaliana accessions to sub-zero temperatures after cold acclimation. Plant Mol Biol 87(1–2):1–15

  27. Leivar P, Monte E (2014) PIFs: systems integrators in plant development. Plant Cell 26(1):56–78. https://doi.org/10.1105/tpc.113.120857

  28. Leivar P, Quail PH (2011) PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci 16(1):19–28. https://doi.org/10.1016/j.tplants.2010.08.003

  29. Li G, Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor Appl Genet 103:455–461

  30. Li G, Gao M, Yang B, Quiros C (2003) Gene for gene alignment between the Brassica and Arabidopsis genomes by direct transcriptome mapping. Theor Appl Genet 107(1):168–180

  31. Mao C, Yi K, Yang L, Zheng B, Wu Y, Liu F, Wu P (2004) Identification of aluminium-regulated genes by cDNA-AFLP in rice (Oryza sativa L.): aluminium-regulated genes for the metabolism of cell wall components. J Exp Bot 55(394):137–143. https://doi.org/10.1093/jxb/erh030

  32. Martelotto LG, Ortiz JPA, Stein J, Espinoza F, Quarin CL, Pessino SC (2005) A comprehensive analysis of gene expression alterations in a newly synthesized Paspalum notatum autotetraploid. Plant Sci 169(1):211–220. https://doi.org/10.1016/j.plantsci.2005.03.015

  33. McCartney AJ, Zhang Y, Weisman LS (2014) Phosphatidylinositol 3,5-bisphosphate: low abundance, high significance. BioEssays 36(1):52–64. https://doi.org/10.1002/bies.201300012

  34. McKenzie JS, Paquin R, Duke SH (1988) Cold and heat tolerance. In: Hanson AA, Barnes DK, Hill RR Jr (eds) Agronomy, vol 29. Madison, WI, pp 259–302

  35. Miao Z, Xu W, Li D, Hu X, Liu J, Zhang R, Tong Z, Dong J, Su Z, Zhang L, Sun M, Li W, Du Z, Hu S, Wang T (2015) De novo transcriptome analysis of Medicago falcata reveals novel insights about the mechanisms underlying abiotic stress-responsive pathway. BMC Genom 16(1):818

  36. Olien CR (1984) An adaptive response of rye to freezing. Crop Sci 24(1):51–54

  37. Olsen JE (2010) Light and temperature sensing and signaling in induction of bud dormancy in woody plants. Plant Mol Biol 73(1–2):37–47. https://doi.org/10.1007/s11103-010-9620-9

  38. Peng X, Wu Q, Teng L, Tang F, Pi Z, Shen S (2015) Transcriptional regulation of the paper mulberry under cold stress as revealed by a comprehensive analysis of transcription factors. BMC Plant Biol 15(1):108. https://doi.org/10.1186/s12870-015-0489-2

  39. Sakai A, Larcher W (1987) Frost survival of plants. Springer, Berlin. c1987. Ecological studies; vol 62. ISBN 3540173323. 0387173323 (U.S.)

  40. Song L, Jiang L, Chen Y, Shu Y, Bai Y, Guo C (2016) Deep-sequencing transcriptome analysis of field-grown Medicago sativa L. crown buds acclimated to freezing stress. Funct Integr Genom 16(5):495–511

  41. Tenhaken R (2015) Cell wall remodeling under abiotic stress. Front Plant Sci 5:771

  42. Volenec JJ, Cunningham SM, Haagenson DM, Berg WK, Joern BC, Wiersma DW (2002) Physiological genetics of alfalfa improvement: past failures, future prospects. Field Crops Res 75(2–3):97–110

  43. Wilson ZN, Scott AL, Dowell RD, Odorizzi G (2018) PI (3, 5) P2 controls vacuole potassium transport to support cellular osmoregulation. Mol Biol Cell 29(14):1718–1731

  44. Wisser RJ, Balint-Kurti PJ, Holland JB (2011) A novel genetic framework for studying response to artificial selection. Plant Genet Resour 9(02):281–283. https://doi.org/10.1017/s1479262111000359

  45. Xu ZS, Chen M, Li LC, Ma YZ (2011) Functions and application of the AP2/ERF transcription factor family in crop improvement. J Integr Plant Biol 53(7):570–585. https://doi.org/10.1111/j.1744-7909.2011.01062.x

  46. Yuan X, Zhang S, Qing X, Sun M, Liu S, Su H, Shu H, Li X (2013) Superfamily of ankyrin repeat proteins in tomato. Gene 523(2):126–136. https://doi.org/10.1016/j.gene.2013.03.122

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Funding

Funding was supported by Competitive grant program of Agriculture and Agri-Food Canada (Grant No. Project 1759).

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Correspondence to Solen Rocher.

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Supplementary File 1 Sequences of forward and reverse SRAP primers used for SRAP-cDNA amplifications (DOCX 18 kb)

Supplementary File 2 Sequences obtained from sequencing of SRAP-cDNA amplicons. Fragment number corresponds to transcript-derived fragment (TDF) listed in Table 1 “fragment ID” column. Consensus (“contig”) sequences are provided when available. Forward or reverse sequences only are provided when one of both strands yielded sequences of low quality (TXT 25 kb)

Supplementary File 3 List of forward and reverse primers used for RT-qPCR amplification of SRAP-cDNA fragments identified in Table 1 and Additional File 4 and for the amplification of two reference genes (DOCX 15 kb)

Supplementary File 4 Results of electrophoresis of SRAP-cDNA fragments amplified with bulked cDNA samples from 50 plants either non-acclimated (NA, 0 h) or cold-acclimated (A) for 8 h at 2 °C and from 50 plants hardened two additional weeks at the non-lethal freezing temperature of − 2 °C (HF). PCR amplification of samples from the alfalfa cultivar Apica (ATF0) and population ATF5 obtained after five cycles of recurrent selection for freezing tolerance within that initial cultivar were performed with SRAP primers listed in Supplementary File 1. Numbers to the left and right of the gels refer to transcript-derived fragments (TDF) ID and corresponds to fragment ID in Table 1 and Supplementary File 2. Molecular weight ladder (MW) and functional annotations of each TDF based on sequence homology with M. truncatula reference genome are included in each gel (PPTX 6362 kb)

Supplementary File 5 Relative levels of expression of 23 SRAP-cDNA transcript-derived fragments (TDF) differentially expressed between crowns of alfalfa non-acclimated (0 h) and crowns cold-acclimated 8 h, 1 d, 3 d, 7 d, 15 d at 2 °C or hardened two additional weeks at the non-lethal freezing temperature of − 2 °C (HF). RT-qPCR amplifications were performed with samples from the alfalfa cultivars Apica (ATF0) and Evolution (ETF0) and populations ATF5 and ETF5 obtained after five cycles of recurrent selection for freezing tolerance in these two initial backgrounds. Means of five pots (10 plants pot). Gene expression was expressed relative to the mean of all samples within each population. Transcript-derived fragments (TDF) were grouped according to their expression profile as: A) Rapidly down regulated; B) Progressively down regulated; C) Transiently up regulated; D) Progressively up regulated. For each population, means with different letters are statistically different at P 0.05. Within each genetic background statistically significant response to recurrent selection at P 0.05, P 0.01 and P 0.001 are respectively indicated by *, ** and *** next to each acclimation treatment (XLSX 33 kb)

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Castonguay, Y., Rocher, S., Bertrand, A. et al. Identification of transcripts associated with the acquisition of superior freezing tolerance in recurrently-selected populations of alfalfa. Euphytica 216, 27 (2020). https://doi.org/10.1007/s10681-020-2559-2

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Keywords

  • Freezing tolerance
  • SRAP-cDNA
  • RT-qPCR
  • Recurrent selection
  • Bulk segregant analysis
  • Alfalfa